 |
EMBO reports 1, 3, 282–286 (2000)
doi:10.1093/embo-reports/kvd045
Published online: September 2000
Figure 2
The EXT1/EXT2 tumor suppressors: catalytic activities and role in heparan sulfate biosynthesis
Claire Senay, Thomas Lind, Kumi Muguruma, Yuko Tone, Hiroshi Kitagawa, Kazuyuki Sugahara, Kerstin Lidholt, Ulf Lindahl & Marion Kusche-Gullberg
| |
| |
|
| |
|
| | |
| |
 |
Glycosyltransferase assays of yeast transformed with EXT proteins. The GlcNAc-T activities of yeast cell extracts were determined by incubating 70  g lysate protein with 0.125 Ci of UDP-[14C]GlcNAc and 50 g of [GlcA-GlcNAc]n oligosaccharide (the reducing-terminal sugar is modified to an 2,5-anhydromannitol unit) under the conditions described (Lind et al., 1993). After 1 h at 37°C, the mixtures were applied to columns (28  0.5 cm) of Sephadex G-25 in 0.5 M NaCl, 50 mM Tris–HCl, 0.1% Triton X-100, pH 7.4. Effluent fractions were analyzed by scintillation counting. The various samples were derived from yeast transformed with empty plasmid (mock, filled diamond), EXT1 (filled circle), EXT2 (open circle) and EXT1/2 (cotransformation, open diamond). GlcA-T activities were assayed in similar fashion, except that UDP-[3H]GlcA was used as a sugar donor, and GlcNAc-[GlcA-GlcNAc]n oligosaccharides were used as acceptor. The resultant gel chromatograms (not shown) were highly similar to the GlcNAc-T assays shown in the figure.
|
|
|
 previous | next  |
| |
| | |
|
